Lactoferrin in the reduction of circulating cholesterol, vascular inflammation, atherosclerosis and cardiovascular disease

ABSTRACT

The present invention relates to methods of using lactoferrin (LF) to reduce circulating levels of cholesterol and vascular inflammation, in order to treat, prevent or reduce the incidence of atherosclerosis and cardiovascular disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional ApplicationNos. 60/498,337 filed Aug. 27, 2003 and 60/430,867 filed Dec. 4, 2002which are incorporated herein by reference.

TECHNICAL FIELD

[0002] The present invention relates to methods of using lactoferrin(LF) to reduce circulating levels of cholesterol and vascularinflammation, in order to treat, prevent or reduce the incidence ofatherosclerosis and cardiovascular disease. More particularly, thepresent invention relates to methods of reducing circulating levels ofcholesterol and vascular inflammation by administering a composition oflactoferrin.

BACKGROUND OF THE INVENTION

[0003] According to current estimates, 61,800,000 people in America haveone or more forms of cardiovascular disease. These diseases claimed958,775 lives in 1999 (40.1 percent of all deaths). Atherosclerosis is aleading form of cardiovascular disease, which involves the slow build-upof fatty plaques on the arterial wall. This build-up can damage thevascular endothelium causing inflammation, a narrowing of the arteriesand potential arterial blockages that can result in heart attacks.Atherosclerosis is a complex disease that starts in childhood and oftenprogresses when people grow older. In some people it progresses rapidly,even in their third decade. Elevated levels of cholesterol, inparticular LDL (low-density lipoprotein), and triglycerides in the bloodhave been associated with the development of fatty plaques, which canlead to generalized vascular damage, atherosclerosis and eventuallyheart attack. Atherosclerosis and cardiac disease is also associatedwith increased cardiovascular inflammation, specifically as measured bylevels of circulating C-reactive protein (CRP).

[0004] One key strategy for reducing the risk of atherosclerosis hasbeen to lower the levels of cholesterol in the blood. Cholesterol levelsin many people can be controlled by diet, but for many patients dietchanges alone are insufficient to reduce high cholesterol. In recentyears, cholesterol lowering drugs such as Zocor® (simvastatin) andLipitor® (atorvastatin) have been increasingly prescribed to helppatients lower their cholesterol levels. These drugs however, are notequally effective in all patients and frequently are associated withsignificant adverse side effects. A second key emerging strategy is thereduction of CRP, an important indicator of vascular inflammation andindependently associated with increased risk of cardiovascular disease.Thus, safer and more effective treatments for lowering cholesterol andfor reducing the vascular inflammation associated with atherosclerosisare of great potential value.

[0005] Lactoferrin is a single chain metal binding glycoprotein. Manycell types, such as monocytes, macrophages, lymphocytes, andbrush-border cells in the intestine, are known to have lactoferrinreceptors. Lactoferrin is found mainly in external secretions of mucosalepithelia such as breast milk, saliva, tears, bile, and pancreatic fluidand has a wide array of functions related to host primary defensemechanisms. For example, lactoferrin has been reported to activatenatural killer (NK) cells, induce colony stimulating activity, activatepolymorphonuclear neutrophils (PMN), regulate granulopoeisis, enhanceantibody-dependent cell cytotoxicity, stimulate lymphokine-activatedkiller (LAK) cell activity, and potentiate macrophage toxicity.

[0006] Recombinant human lactoferrin has previously been described asbeing purified after expression in a variety of prokaryotic andeukaryotic organisms including aspergillus (U.S. Pat. No. 6,080,559),cattle (U.S. Pat. No. 5,919,913), rice, corn, Sacharomcyes (U.S. Pat.No. 6,228,614) and Pichia pastoris (U.S. Pat. Nos. 6,455,687, 6,277,817,6,066,469). Also described are expression systems for the expression offull-length human lactoferrins (e.g., U.S. Pat. No. 6,100,054). In allcases, part of the teaching is expression of the full length cDNA andpurification of the intact protein whose N-terminal, after processing ofthe leader peptide, is the amino acid glycine. Nuijens et al. (U.S. Pat.No. 6,333,311) separately describe variants of human lactoferrin buttheir focus is limited to deletion or substitution of arginine residuesfound in the N-terminal domain of lactoferrin.

[0007] The present invention is the first to use a lactoferrincomposition as a means of reducing cholesterol and cardiovascularinflammation and for treating or reducing atherosclerosis andcardiovascular disease.

BRIEF SUMMARY OF THE INVENTION

[0008] The present invention is directed to a method for modulatingcirculating levels of cholesterol and reducing the vascular inflammationassociated with atherosclerosis and cardiovascular disease. The methodof treatment involves administration of a lactoferrin composition.

[0009] The lactoferrin composition, which is dispersed in apharmaceutically acceptable carrier, comprises lactoferrin or anN-terminal lactoferrin variant in which at least the N-terminal glycineresidue is truncated or substituted. The lactoferrin is mammalianlactoferrin, more particularly, the lactoferrin is human or bovine. Yetfurther, the lactoferrin is recombinant lactoferrin. N-terminallactoferrin variants include variants that at least lack the N-terminalglycine residue or contain a substitution at the N-terminal glycineresidue. The substitution can comprise substituting a natural orartificial amino acid residue for the N-terminal glycine residue. Forexample, the substitution can comprise substituting a positive aminoacid residue or a negative amino acid residue for the N-terminal glycineresidue or substituting a neutral amino acid residue other than glycinefor the N-terminal glycine residue. Other N-terminal lactoferrinvariants include lactoferrin lacking one or more N-terminal residues orhaving one or more substitutions in the N-terminal. In specificembodiments, the N-terminal lactoferrin variant comprises at least 1% ofthe lactoferrin composition, at least 5% of the lactoferrin composition,at least 10% of the lactoferrin composition, at least 25% of thelactoferrin composition, at least 50% of the lactoferrin composition orany range in between.

[0010] The amount of the lactoferrin that is administered is about 1 ngto about 20 g per day, more preferably, the amount is about 0.1 g toabout 5 g per day. More particularly, the composition is a solution,capsule or a tablet having a lactoferrin concentration of about 0.1% toabout 100%.

[0011] In further embodiments, a metal chelator dispersed in apharmaceutically acceptable carrier can also be administered with thelactoferrin composition. Preferred metal chelator include, but are notlimited to ethylenediaminetetraacetic acid (EDTA) or[ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA). Morepreferably, the metal chelator is EDTA. The amount of EDTA that isadministered is about 1 ng to about 1 g per day.

[0012] An embodiment of the present invention is a method of treating acardiovascular disease comprising the step of administering to a subjecta lactoferrin composition in an effective amount to provide animprovement in the cardiovascular disease, for example, atherosclerosis.The lactoferrin composition reduces the levels of circulating totalcholesterol, low density lipoproteins (LDL), or very low densitylipoproteins (VLDL). Still further, the lactoferrin compositionincreases the levels of circulating high density lipoproteins (HDL). Inaddition to modulating levels of cholesterol, the lactoferrincomposition reduces the levels of vascular inflammation, circulatingC-reactive protein (CRP), triglycerides, or proliferation of vascularsmooth muscle cells. The lactoferrin composition may also reducevascular spasms or vascular hyper-reactivity or promote endothelialintegrity or healing. In further embodiments, the lactoferrincomposition reduces the production or activity of pro-inflammatorycytokines.

[0013] The lactoferrin composition of the present invention can beadministered parenterally, for example, subcutaneously, intramuscularly,intraperitoneally, intravenously, intraarterially, intramyocardially,transendocardially, transepicardially, or intrathecally.

[0014] In a further embodiment, the lactoferrin composition isadministered orally. For oral administration, an antacid in combinationwith the lactoferrin composition can be administered. The lactoferrincan be formulated in a delayed release formulation. Still further, thelactoferrin composition can be formulated wherein release occurs in thesmall intestine or in the large intestine.

[0015] Another embodiment of the present invention is a method ofmodulating atherosclerosis in a subject comprising the step ofadministering to the subject a lactoferrin composition in an effectiveamount to modulate atherosclerosis in the subject. Modulating isreducing the incidence of atherosclerosis or reducing the severity ofatherosclerosis. In further embodiments, the lactoferrin composition canbe administered in combination with an anti-cholesterol agent or ananti-inflammatory agent. The anti-cholesterol agent is selected from thegroup consisting of cholesterol absorption inhibitors, bile acidsequestrants (cholestryramine, cholestipol and colesevalam), nicotinicacid, fibric acids (gemfibrozil, fenofibrate and clofibrate) and HMG-coAreductase inhibitors (lovastatin, pravastatin, simvastatin, fluvastatin,atorvastatin and cerivastatin).

[0016] Another embodiment is a method of preventing a cardiovasculardisease in a subject at risk for developing a cardiovascular diseasecomprising the step of administering to the subject a lactoferrincomposition in an amount sufficient to result in prophylaxis of thecardiovascular disease in the subject. The cardiovascular disease isatherosclerosis.

[0017] Still further, another embodiment is a method of reducing therisk of cardiovascular disease in a subject at risk for developing acardiovascular disease comprising the step of administering to thesubject a lactoferrin composition in an effective amount to result in areduction of risk of the cardiovascular disease in the subject. Thecardiovascular disease is atherosclerosis.

[0018] The foregoing has outlined rather broadly the features andtechnical advantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated that the conception and specific embodimentdisclosed may be readily utilized as a basis for modifying or designingother structures for carrying out the same purposes of the presentinvention. It should also be realized that such equivalent constructionsdo not depart from the invention as set forth in the appended claims.The novel features which are believed to be characteristic of theinvention, both as to its organization and method of operation, togetherwith further objects and advantages will be better understood from thefollowing description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] For a more complete understanding of the present invention,reference is now made to the following descriptions taken in conjunctionwith the accompanying drawings.

[0020]FIG. 1 shows the net reduction of total serum cholesterol insubjects receiving oral rhLF for seven days with respect to patientsreceiving placebo for seven days.

[0021]FIG. 2 shows the reduction of C-reactive protein in subjectsreceiving oral rhLF for seven days.

[0022]FIG. 3A and FIG. 3B show the effect of rhLF on hyperlipidemia inmice. FIG. 3A shows the effect of rhLF on HDL, LDL and total cholesterollevels. FIG. 3B shows the effect on HDL/LDL ratio.

[0023]FIG. 4 shows a dose effect of rhLF on hyperlipidemia in mice.

[0024]FIG. 5 shows the effect of oral rhLF with lovastatin onhyperlipidemia in mice.

DETAILED DESCRIPTION OF THE INVENTION

[0025] It is readily apparent to one skilled in the art that variousembodiments and modifications can be made to the invention disclosed inthis Application without departing from the scope and spirit of theinvention.

[0026] As used herein, the use of the word “a” or “an” when used inconjunction with the term “comprising” in the claims and/or thespecification may mean “one,” but it is also consistent with the meaningof “one or more,” “at least one,” and “one or more than one.” Stillfurther, the terms “having”, “including”, “containing” and “comprising”are interchangeable and one of skill in the art is cognizant that theseterms are open ended terms.

[0027] The term “atherosclerosis” as used herein includes a form ofarteriosclerosis characterized by a combination of changes in the intimaof arteries, such changes include, but are not limited to accumulationof lipids, complex carbohydrates, blood and blood products, fibroustissue and calcium deposits. Yet further, atherosclerotic plaques can becharacterized into at least two areas. One type is characterized byprominent proliferation of cells with small accumulations of lipids. Thesecond type consists mostly of intracellular and extracellular lipidaccumulation and a small amount of cellular proliferation.

[0028] The term “cardiovascular disease or disorder” as used hereinrefers to disease and disorders related to the cardiovascular orcirculatory system. Cardiovascular disease and/or disorders include, butare not limited to, diseases and/or disorders of the pericardium, heartvalves (e.g., incompetent valves, stenosed valves, rheumatic heartdisease, mitral valve prolapse, aortic regurgitation), myocardium (e.g.,coronary artery disease, myocardial infarction, heart failure, ischemicheart disease, angina) blood vessels (e.g., hypertension,arteriosclerosis, aneurysm) or veins (e.g., varicose veins,hemorrhoids). Yet further, one skill in the art recognizes thatcardiovascular diseases and/or disorders can result from congenitaldefects, genetic defects, environmental influences (e.g., dietaryinfluences, lifestyle, stress, etc.), and other defects or influences.

[0029] The term “chemokine” as used herein refers to small cytokinesthat are involved in the migration and activation of cells, for examplephagocytic cells and lymphocytes. One of skill in the art realizes thatchemokines play a central role in inflammatory and immune responseprocesses.

[0030] The term “cholesterol” as used herein refers to the monohydricalcohol form, which is a white, powdery substance that is found in allanimal cells and in animal-based foods (not in plants). Cholesterol isan essential nutrient necessary for many functions, including thefollowing: repairing cell membranes, manufacturing vitamin D on theskin's surface, production of hormones, such as estrogen andtestosterone, and possibly helping cell connections in the brain thatare important for learning and memory.

[0031] The term “chylomicron” as used herein refers to the largest insize and lowest in density of the triglyceride carrying lipoproteins.

[0032] The term “cytokine” as used herein refers to proteins that aremade by cells that affect the behavior of other cells, for examplestimulate or inhibit cell proliferation. For example, cytokines that aremade by lymphocytes are often called lymphokines or interleukins. One ofskill in the art realizes that the term cytokine is a generic term usedin the literature to refer to proteins that are made by cells that caneffect the behavior of other cells.

[0033] The term “effective amount” or “therapeutically effective amount”are interchangeable as used herein and refer to an amount that resultsin an improvement or remediation of the symptoms of the disease orcondition.

[0034] The term “high-density lipoprotein” or “HDL” as used herein isthe smallest and most dense type of cholesterol-carrying lipoprotein andis often referred to as the “good” cholesterol.

[0035] The term “intermediate density lipoprotein” or “IDL” as usedherein refers to a triglyceride-carrying lipoprotein.

[0036] The term “lactoferrin” or “LF” as used herein refers to native orrecombinant lactoferrin. Native lactoferrin can be obtained bypurification from mammalian milk or colostrum or from other naturalsources. Recombinant lactoferrin (rLF) can be made by recombinantexpression or direct production in genetically altered animals, plants,fungi, bacteria, or other prokaryotic or eukaryotic species, or throughchemical synthesis.

[0037] The term “lactoferrin composition” as used herein refers to acomposition having lactoferrin, a portion or part of lactoferrin, anN-terminal lactoferrin variant, or a combination thereof.

[0038] The term “lipid” as used herein refers to the building blocks ofany of the fats or fatty substances found in animals and plants, whichare characterized by their insolubility in water and solubility in fatsolvents such as alcohol, ether and chloroform. Lipids include fats(e.g., esters of fatty acids and glycerol); lipoids (e.g.,phospholipids, cerebrosides, waxes) and sterols (e.g., cholesterol).

[0039] The term “lipoproteins” as used herein are protein spheres thattransport cholesterol, triglyceride, or other lipid molecules throughthe bloodstream. Lipoproteins are categorized into five types accordingto size and density. They can be further defined by whether they carrycholesterol [the two smaller lipoproteins (HDL and LDL)] ortriglycerides [the three largest lipoproteins (IDL, VLDL, andchylomicrons)].

[0040] The term “low density lipoprotein” or “LDL” as used herein is atype of cholesterol-carrying lipoprotein which is often called the “bad”cholesterol.

[0041] The term “N-terminal lactoferrin variant” as used herein refersto lactoferrin wherein at least the N-terminal glycine has beentruncated and/or substituted. N-terminal lactoferrin variants alsoinclude, but are not limited to deletion and/or substitution of one ormore N-terminal amino acid residues, for example 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 16 N-terminal amino acid residues, etc.Thus, N-terminal lactoferrin variants comprise at least deletions ortruncations and/or substitutions of 1 to 16 N-terminal amino acidresidues. The deletion and/or substitution of at least the N-terminalglycine of lactoferrin mediates the same biological effects asfull-length lactoferrin and/or may enhance lactoferrin's biologicalactivity, for example by stimulating the production of various cytokines(e.g., IL-18, MIP-3α, GM-CSF or IFN-γ) by inhibiting various cytokines,(e.g., IL-2, IL-4, IL-5, IL-10, or TNF-α) by improving a cardiovasculardisease, e.g., atherosclerosis, or the parameters relating tocardiovascular disease including circulating levels of totalcholesterol, HDL, LDL, VLDL, trigylcerides and C-reactive protein (CRP).

[0042] The term “metal chelator” as used herein refers to a compoundwhich binds metal. Metal chelators that can be used in the presentinvention include the divalent metal chelators, for example,ethylenediaminetetraacetic acid (EDTA), [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA),1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),hydroxyethlene triamine diacetic acid, (HEDTA) or salts thereof.

[0043] The term “oral administration” as used herein includes oral,buccal, enteral or intragastic administration.

[0044] The term “parenteral administration” as used herein includes anyform of administration in which the compound is absorbed into thesubject without involving absorption via the intestines. Exemplaryparenteral administrations that are used in the present inventioninclude, but are not limited to intramuscular, intravenous,intraperitoneal, intraocular, subcutaneous or intraarticularadministration. Yet further, parenteral administration also includesadministration into a surgical field.

[0045] The term “pharmaceutically acceptable carrier” as used hereinincludes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the vectors or cells of the presentinvention, its use in therapeutic compositions is contemplated.Supplementary active ingredients also can be incorporated into thecompositions.

[0046] The term “preventing” as used herein refers to minimizing,reducing or suppressing the risk of developing a disease state orparameters relating to the disease state or progression or otherabnormal or deleterious conditions.

[0047] The term “statin” as used herein includes compounds that areHMG-CoA reductase inhibitors, for example, but not limited tosimvastatin (Zocor®) and atorvastatin (Lipitor®). Thus, as used hereinthe terms “statin” and “HMG-CoA reductase inhibitor” areinterchangeable.

[0048] The term “subject” as used herein, is taken to mean any mammaliansubject to which a lactoferrin composition is administered according tothe methods described herein. Thus, a skilled artisan realizes that amammalian subject, includes, but is not limited to humans, monkeys,horses, pigs, cows, dogs, cats, rats and mice. In a specific preferredembodiment, the methods of the present invention are employed to treat ahuman subject. In more preferred embodiments, the subject has signs orindicators of atherosclerosis. These signs or indicators include, forexample, the development of cholesterol plaques in the arteries andcalcification, the extent of which can be determined by Sudan IVstaining, or the development of foam cells in an artery or arterialspasm. Atherosclerosis also is characterized by a narrowing of thearteries detected by, for example, coronary angioplasty, ultrasound andultrafast CT. In further embodiments, the subject is at risk ofdeveloping a cardiovascular disease. Thus, the subject may or may not becognizant of their disease state or potential disease state and may ormay not be aware that they are need of treatment (therapeutic treatmentor prophylactic treatment).

[0049] The term “topical administration” as used herein includes, but isnot limited to topical, dermal, or epidermal.

[0050] The term “total cholesterol” as used herein refers to the sum ofthree kinds of lipids: high-density lipoprotein (HDL), low-densitylipoprotein (LDL), and triglycerides. Levels of serum total cholesterolof >200 mg/dl are levels that are an indicating risk factor foratherosclerosis and cardiovascular disease.

[0051] The term “triglycerides” as used herein are composed of fattyacid molecules and are the basic chemicals contained in fats in bothanimals and plants.

[0052] The term “treating” and “treatment” as used herein refers toadministering to a subject a therapeutically effective amount of arecombinant human lactoferrin composition so that the subject has animprovement in a cardiovascular disease or the parameters relating tocardiovascular disease including circulating levels of totalcholesterol, HDL, LDL, VLDL, trigylcerides and C-reactive protein (CRP).The improvement is any observable or measurable improvement. Thus, oneof skill in the art realizes that a treatment may improve the patientcondition, but may not be a complete cure of the disease.

[0053] The term “very low density lipoprotein” or “VLDL” as used hereinrefers to a triglyceride carrying lipoprotein.

[0054] A. Lactoferrin

[0055] The lactoferrin used according to the present invention can beobtained through isolation and purification from natural sources, forexample, but not limited to mammalian milk. The lactoferrin ispreferably mammalian lactoferrin, such as bovine or human lactoferrin.In preferred embodiments, the lactoferrin is produced recombinantlyusing genetic engineering techniques well known and used in the art,such as recombinant expression or direct production in geneticallyaltered animals, plants or eukaryotes, or chemical synthesis. See, forexample, U.S. Pat. Nos. 5,571,896; 5,571,697 and 5,571,691, which areherein incorporated by reference.

[0056] In certain aspects, the present invention provides lactoferrinvariants having enhanced biological activities over natural LF and orrLF, e.g., the ability to stimulate and/or inhibit cytokines orchemokines. In particular, the invention provides variants oflactoferrin from which at least the N-terminal glycine residue has beensubstituted and/or truncated. The N-terminal lactoferrin variants mayoccur naturally or may be modified by the substitution or deletion ofone or more amino acids.

[0057] The deletional variants can be produced by proteolysis oflactoferrin and/or expression of a polynucleotide encoding a truncatedlactoferrin as described in U.S. Pat. No. 6,333,311, which isincorporated herein by reference.

[0058] Substitutional variants or replacement variants typically containthe exchange of one amino acid for another at one or more sites withinthe protein. Substitutions can be conservative, that is, one amino acidis replaced with one of similar shape and charge. Conservativesubstitutions are well known in the art and include, for example, thechanges of: alanine to serine; arginine to lysine; asparagine toglutamine or histidine; aspartate to glutamate; cysteine to serine;glutamine to asparagine; glutamate to aspartate; glycine to proline;histidine to asparagine or glutamine; isoleucine to leucine or valine;leucine to valine or isoleucine; lysine to arginine; methionine toleucine or isoleucine; phenylalanine to tyrosine, leucine or methionine;serine to threonine; threonine to serine; tryptophan to tyrosine;tyrosine to tryptophan or phenylalanine; and valine to isoleucine orleucine.

[0059] In making such changes, the hydropathic index of amino acids maybe considered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982). It is accepted thatthe relative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

[0060] Each amino acid has been assigned a hydropathic index on thebasis of their hydrophobicity and charge characteristics (Kyte andDoolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

[0061] It is known in the art that certain amino acids may besubstituted by other amino acids having a similar hydropathic index orscore and still result in a protein with similar biological activity,e.g., still obtain a biological functionally equivalent protein. Inmaking such changes, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those that are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

[0062] It is also understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity. U.S.Pat. No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine(−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5);tryptophan (−3.4).

[0063] Still further, it is understood that an amino acid can besubstituted for another having a similar hydrophilicity value and stillobtains a biologically equivalent and immunologically equivalentprotein. In such changes, the substitution of amino acids whosehydrophilicity values are within ±2 is preferred, those that are within±1 are particularly preferred, and those within ±0.5 are even moreparticularly preferred.

[0064] Thus, in the present invention, substitutional variants orreplacement can be produced using standard mutagenesis techniques, forexample, site-directed mutagenesis as disclosed in U.S. Pat. Nos.5,220,007; 5,284,760; 5,354,670; 5,366,878; 5,389,514; 5,635,377;5,789,166, and 6,333,311, which are incorporated herein by reference. Itis envisioned that at least the N-terminal glycine amino acid residuecan be replaced or substituted with any of the twenty natural occurringamino acids, for example a positively charged amino acid (arginine,lysine, or histidine), a neutral amino acid (alanine, asparagine,cysteine, glutamine, glycine, isoleucine, leucine, methionine,phenylaline, proline, serine, threonine, tryptophan, tyrosine, valine)and/or a negatively charged amino acid (aspartic acid or glutamic acid).Still further, it is contemplated that any amino acid residue within therange of N1 to N16 can be replaced or substituted. It is envisioned thatat least up to 16 of the N-terminal amino acids residues can be replacedor substituted as long as the protein retains it biological and/orfunctional activity, which is stimulating the production of variouscytokines, (e.g., IL-18, MIP-3α, GM-CSF or IFN-γ) by inhibiting variouscytokines, (e.g., IL-2, IL-4, IL-5, IL-10, and TNF-α) by improving acardiovascular disease, e.g., atherosclerosis, or the parametersrelating to cardiovascular disease including circulating levels of totalcholesterol, HDL, LDL, VLDL, trigylcerides and C-reactive protein (CRP).Thus, the N-terminal lactoferrin variants of the present invention areconsidered functional equivalents of lactoferrin.

[0065] In terms of functional equivalents, it is well understood by theskilled artisan that, inherent in the definition of a “biologicallyfunctional equivalent” protein is the concept that there is a limit tothe number of changes that may be made within a defined portion of themolecule while retaining a molecule with an acceptable level ofequivalent biological activity and/or enhancing the biological activityof the lactoferrin molecule. Biologically functional equivalents arethus defined herein as those proteins in which selected amino acids (orcodons) may be substituted. Functional activity is defined as theability of lactoferrin to stimulate or inhibit various cytokines orchemokines and/or by improving a cardiovascular disease, e.g.,atherosclerosis, or the parameters relating to cardiovascular diseaseincluding circulating levels of total cholesterol, HDL, LDL, VLDL,trigylcerides and C-reactive protein (CRP).

[0066] Still further, the N-terminal amino acid residues can besubstituted with a modified and/or unusual amino acids. A table ofexemplary, but not limiting, modified and/or unusual amino acids isprovided herein below. TABLE 1 Modified and/or Unusual Amino Acids Abbr.Amino Acid Abbr. Amino Acid Aad 2-Aminoadipic acid EtAsnN-Ethylasparagine BAad 3-Aminoadipic acid Hyl Hydroxylysine BAlabeta-alanine, beta-Amino-propionic AHyl allo-Hydroxylysine acid Abu2-Aminobutyric acid 3Hyp 3-Hydroxyproline 4Abu 4-Aminobutyric acid,piperidinic 4Hyp 4-Hydroxyproline acid Acp 6-Aminocaproic acid IdeIsodesmosine Ahe 2-Aminoheptanoic acid Aile allo-Isoleucine Aib2-Aminoisobutyric acid MeGly N-Methylglycine, sarcosine BAib3-Aminoisobutyric acid MeIle N-Methylisoleucine Apm 2-Aminopimelic acidMeLys 6-N-Methyllysine Dbu 2,4-Diaminobutyric acid MeVal N-MethylvalineDes Desmosine Nva Norvaline Dpm 2,2′-Diaminopimelic acid Nle NorleucineDpr 2,3-Diaminopropionic acid Orn Ornithine EtGly N-Ethylglycine

[0067] The presence and the relative proportion N-terminal lactoferrinvariants (deletions and/or substitutions) in a preparation oflactoferrin (lactoferrin composition) may be done by determination ofthe N-terminal amino acid sequence by the process of Edman degradationusing standard methods. A relative proportion of N-terminal lactoferrinvariant comprise at least 1% of the lactoferrin composition, at least 5%of the lactoferrin composition, at least 10% of the lactoferrincomposition, at least 25% of the lactoferrin composition, at least 50%of the lactoferrin composition or any range in between.

[0068] In this method, the protein is reacted with phenylisothiocyanate(PITC), which reacts with the amino acid residue at the amino terminusunder basic conditions to form a phenylthiocarbamyl derivative(PTC-protein). Trifluoroacetic acid then cleaves off the first aminoacid as its anilinothialinone derivative (ATZ-amino acid) and leaves thenew amino terminus for the next degradation cycle.

[0069] The percentage of N-terminal lactoferrin variant may also be donemore precisely by using a Dansylation reaction. Briefly, protein isdansylated using Dansyl chloride reacted with the protein in alkalineconditions (pH 10). Following the Dansylation, the reaction mixtures aredried to pellets, then completely hydrolyzed in 6N HCl. The proportionof N-terminal amino acids are identified by RP HPLC using an in-linefluorometer in comparison with standards made up of known dansylatedamino acids.

[0070] B. Pharmaceutical Compositions

[0071] The present invention is drawn to a composition comprisinglactoferrin that is dispersed in a pharmaceutical carrier, which is usedto treat cardiovascular disease. The lactoferrin that is contained inthe composition of the present invention comprises lactoferrin or anN-terminal lactoferrin variant in which at least the N−1 terminalglycine residue is truncated or substituted. More specifically, theN-terminal lactoferrin variant comprises at least 1% of the composition,at least 5% of the composition, at least 10% of the composition, atleast 25% of the composition, at least 50% of the composition or anyrange in between.

[0072] Yet further, the composition comprises lactoferrin in combinationwith a metal chelator dispersed in a pharmaceutical carrier. Thus, thepresent invention is drawn to a lactoferrin composition with or withouta metal chelator that is dispersed in a pharmaceutical carrier. One ofskill in the art understands that both compositions (e.g., lactoferrinalone or lactoferrin in combination with a metal chelator) are withinthe scope of the present invention and can be used interchangeablydepending upon the type of response that is desired. It is envisionedthat the addition of a metal chelator to the lactoferrin compositionenhances the sequestering of metal ions and thus strengthens the immunesystem or enhances the effect of lactoferrin.

[0073] Metal chelators that can be used in combination with lactoferrin,include the divalent metal chelators, for example,ethylenediaminetetraacetic acid (EDTA),[ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA),1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),hydroxyethlene triamine diacetic acid, (HEDTA) or any salts thereof.More preferably, EDTA is used in combination with lactoferrin.

[0074] Further in accordance with the present invention, the compositionof the present invention suitable for administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, e.g.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent or carrier is detrimental to the recipient or to thetherapeutic effectiveness of a the composition contained therein, itsuse in administrable composition for use in practicing the methods ofthe present invention is appropriate. Examples of carriers or diluentsinclude fats, oils, water, saline solutions, lipids, liposomes, resins,binders, fillers and the like, or combinations thereof.

[0075] In accordance with the present invention, the composition iscombined with the carrier in any convenient and practical manner, e.g.,by solution, suspension, emulsification, admixture, encapsulation,absorption and the like. Such procedures are routine for those skilledin the art.

[0076] In a specific embodiment of the present invention, thecomposition is combined or mixed thoroughly with a semi-solid or solidcarrier. The mixing can be carried out in any convenient manner such asgrinding. Stabilizing agents can be also added in the mixing process inorder to protect the composition from loss of therapeutic activity,e.g., denaturation in the stomach. Examples of stabilizers for use in anthe composition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc., proteolytic enzymeinhibitors, and the like. Yet further, it is envisioned that divalentmetal chelators, for example EDTA, can also be used to stabilize thecomposition of the present invention. More preferably, for an orallyadministered composition, the stabilizer can also include antagonists tothe secretion of stomach acids.

[0077] The composition for oral administration which is combined with asemi-solid or solid carrier can be further formulated into hard or softshell gelatin capsules, tablets, or pills. More preferably, gelatincapsules, tablets, or pills are enterically coated. Enteric coatingsprevent denaturation of the composition in the stomach or upper bowelwhere the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001. Uponreaching the small intestines, the basic pH therein dissolves thecoating and permits the lactoferrin composition to be released andabsorbed by specialized cells, e.g., epithelial enterocytes and Peyer'spatch M cells.

[0078] In another embodiment, a powdered composition is combined with aliquid carrier such as, e.g., water or a saline solution, with orwithout a stabilizing agent.

[0079] The compositions of the present invention may be formulated in aneutral or salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

[0080] Sterile injectable solutions are prepared by incorporating thelactoferrin in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

[0081] Further, the composition for topical administration which iscombined with a semi-solid carrier can be further formulated into a gelointment. A preferred carrier for the formation of a gel ointment is agel polymer. Preferred polymers that are used to manufacture a gelcomposition of the present invention include, but are not limited tocarbopol, carboxymethyl-cellulose, and pluronic polymers. Gel polymersprevent denaturation of the composition in the open skin by serumproteases.

[0082] The amount of lactoferrin in the present invention may vary fromabout 1 ng to about 100 g of lactoferrin, more preferably 1 ng to 20 gper day, most preferably 1 μg to 5 g. In preferred embodiments, thecomposition of the present invention comprises a lactoferrinconcentration of about 0.1% to about 100%. The lactoferrin compositionmay comprise lactoferrin or an N-terminal lactoferrin variant in whichat least the N−1 terminal glycine residue is truncated and/orsubstituted.

[0083] More preferably, the composition of the present invention alsocontains metal chelators, for example, but not limited toethylenediaminetetraacetic acid (EDTA),[ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA),1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),hydroxyethlene triamine diacetic acid, (HEDTA) or salts thereof. Theamount of the metal chelator in the composition may vary from about 1 ngto about 1 g. A preferred metal chelator is EDTA.

[0084] Upon formulation, solutions are administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective to result in an improvement or remediation ofthe symptoms. The formulations are easily administered in a variety ofdosage forms such as ingestible solutions, drug release capsules and thelike. Some variation in dosage can occur depending on the condition ofthe subject being treated. The person responsible for administrationcan, in any event, determine the appropriate dose for the individualsubject.

[0085] C. Treatment or Prophylaxis of Cardiovascular Disease

[0086] In accordance with the present invention, the compositionprovided in any of the above-described pharmaceutical carriers isadministered to a subject who has experienced or is at risk ofdeveloping cardiovascular disease. Risk factors include, but are notlimited to elevated levels of cholesterol or CRP. One of skill in theart can determine the patients who would potentially benefit from atherapeutic agent that would reduce circulating levels of totalcholesterol or triglycerides or cardiovascular inflammation. One ofskill in the art can determine the therapeutically effective amount ofthe composition to be administered to a subject based upon severalconsiderations, such as local effects, pharmacodynamics, absorption,metabolism, method of delivery, age, weight, disease severity andresponse to the therapy.

[0087] Oral administration of the composition includes oral, buccal,enteral or intragastric administration. It is also envisioned that thecomposition may be used as a food additive. For example, the compositionis sprinkled on food or added to a liquid prior to ingestion.

[0088] In addition to oral administration, the lactoferrin compositioncan also be administered parenterally, which includes, but is notlimited to intradermal, subcutaneous, intramuscular, intraperitoneal,intravenous, intraarterial, intramyocardial, transendocardial,transepicardial, intrathecal, and infusion techniques.

[0089] Cardiovascular diseases and/or disorders include, but are notlimited to, diseases and/or disorders of the pericardium, heart valves(e.g., incompetent valves, stenosed valves, Rheumatic heart disease,mitral valve prolapse, aortic regurgitation), myocardium (e.g., coronaryartery disease, myocardial infarction, heart failure, ischemic heartdisease, angina) blood vessels (e.g., hypertension, arteriosclerosis,aneurysm) or veins (e.g., varicose veins, hemorrhoids). In specificembodiments, the cardiovascular disease is atherosclerosis.

[0090] In specific embodiments of the present invention, the lactoferrincomposition is administered to a subject suffering from or at risk fordeveloping atherosclerosis. Thus, it is envisioned that the lactoferrincomposition modulates or reduces the severity and/or incidence ofatherosclerosis.

[0091] Prophylactic treatment can be administered to those subjects atrisk for developing atherosclerosis. One risk factor is an atherogeniclipoprotein profile. For example, a ratio of serum cholesterol to highdensity lipoproteins of above 5:1 indicates a higher than average riskof developing atherosclerosis. Other factors indicating increased riskfor atherosclerosis include a serum cholesterol level of above 240mg/dl; a high density lipoprotein level below about 35 mg/dl; and a lowdensity lipoprotein level above about 160 mg/dl.

[0092] Another embodiment includes treating a human subject with anelevated level of circulating total cholesterol or CRP according to thethen medically established guidelines. It is contemplated that thelactoferrin composition of the present invention reduces or attenuatesthe levels of circulating total cholesterol, low density lipoproteins orvery low density lipoproteins. It is contemplated that the lactoferrincomposition of the present invention can interfere with how cholesterolenters the circulation either via absorption from food (exogenouspathway) or synthesis by the liver (endogenous pathway).

[0093] In further embodiments, the composition is administered inconjunction with an antacid. Thus, an antacid is administered prior orsubstantially simultaneously with or after oral administration of thecomposition. The administration of an antacid just prior or immediatelyfollowing the administration of the composition may help to reduce thedegree of inactivation of the lactoferrin in the digestive tract.Examples of appropriate antacids include, but are not limited to, sodiumbicarbonate, magnesium oxide, magnesium hydroxide, calcium carbonate,magnesium trisilicate, magnesium carbonate and alumin hydroxide gel.

[0094] In a preferred embodiment of the present invention, thecomposition is administered in an effective amount to decrease, reduce,inhibit or abrogate cardiovascular disease. Thus, a subject isadministered a therapeutically effective amount of a lactoferrincomposition so that the subject has an improvement in the parametersrelating to cardiovascular disease including circulating levels of totalcholesterol, HDL, LDL, VLDL, trigylcerides and C-reactive protein (CRP).The amount of lactoferrin in the composition may vary from about 1 ng toabout 20 g. Preferably, the composition that is orally administeredcontains the range of 0.5 g to 5 g of lactoferrin per day.

[0095] The improvement is any observable or measurable improvement.Thus, one of skill in the art realizes that a treatment may improve thepatient condition, but may not be a complete cure of the disease. Incertain aspects, the composition is administered in an effective amountto decrease, reduce, inhibit or abrogate excess amounts of cholesterollevels in circulation. A subject requires treatment for cholesterollevels based upon any of the following situations: LDL of 160 mg/ml orgreater; LDL of 130-159 mg/ml and also have two or more cardiovascularrisk factors; LDL of 100 mg/ml or greater in subjects with coronaryheart disease (CHD); triglycerides of 200 mg/dl or higher; totalcholesterol of 240 mg/dl or higher or HDL of less than 40 mg/dl. Thus,after administration of lactoferrin, if any of the above conditionsimprove, then the amount of lactoferrin is considered an effectiveamount to decrease, reduce, inhibit or abrogate cholesterol levels inthe circulation.

[0096] Another embodiment is a method of reducing vascular inflammationby administering the lactoferrin composition of the present invention.Vascular inflammation can be tied to a number of the underlyingprocesses contributing to atherosclerosis which include endothelialdysfunction, vascular proliferation and matrix alteration. Recentstudies have emphasized the involvement of inflammation in mediating allstages of atherosclerosis. Vascular inflammation is thought to be aconsequence of damage to the vascular endothelium and may also involvethe proliferation of vascular smooth muscle cells (vsmcs). One precursorof lesion development in humans may be focal accumulation of vsmcswithin the intima. In early atherosclerosis, vsmcs may contribute to thedevelopment of the atheroma through the production of pro-inflammatorymediators such as monocyte chemoattractant protein 1 and vascular celladhesion molecule, and through the synthesis of matrix moleculesrequired for the retention of lipoproteins. Inflammation of the vascularendothelium and proliferation of vsmcs may also impact the stability ofthe plaque through the formation of a firm fibrous cap. Indeed, inlipid-laden lesions in which the fibrous cap is thin and weak, there isevidence of vsmc apoptosis, especially at the “shoulder” region,associated with inflammation. In addition, the local inflammatory milieucan induce expression of collagenase and inhibit expression ofproteolytic inhibitors, thus rendering the fibrous cap weak andsusceptible to rupture. Lactoferrin, having known anti-inflammatoryproperties, may thus serve to inhibit the underlying processesassociated with the development of atherosclerosis.

[0097] In further embodiments, the lactoferrin composition may alsoreduce vascular spasms or vascular hyper-reactivity. Vascular spasms area sudden, brief tightening of a blood vessel, which can temporarilyreduce blood flow to tissues supplied by that vessel.

[0098] Still further, the lactoferrin composition may also promoteendothelial integrity or healing. Endothelia are the layer of cellslining the blood vessels. Endothelial dysfunction most commonly refersto impairment of endothelium-dependent vasodilation and widespreadabnormalities in endothelial integrity and homeostasis. It is believedthat HDLs help maintain endothelial integrity, facilitate vascularrelaxation, inhibit blood cell adhesion to vascular endothelium, reduceplatelet aggregability and coagulation, and may favor fibrinolysis. Theintegrity or completeness of the endothelia lining of the vessels isimportant to preventing/treating the development of plaques andatherosclerosis. Thus, it is envisioned that the lactoferrin compositionof the present invention will promote or modulate endothelial integrityor healing.

[0099] Yet further, another embodiment is a method of preventing acardiovascular disease in a subject at risk for developing acardiovascular disease comprising the step of administering to thesubject a lactoferrin composition in an amount sufficient to result inprophylaxis of the cardiovascular disease in the subject. In preferredembodiments, the cardiovascular disease is atherosclerosis. It isenvisioned that the lactoferrin composition not only possess therapeuticbenefits for those subjects suffering from cardiovascular diseases, butalso possess prophylactic properties for those subjects at risk fordeveloping cardiovascular disease. A subject at risk may or may not becognizant of their disease state or potential disease state and may ormay not be aware that they are need of treatment.

[0100] Thus, prophylatically, it is envisioned that the lactoferrincomposition can reduce any of the following: the levels of circulatingtotal cholesterol, low density lipoproteins (LDL), very low densitylipoproteins (VLDL), levels of vascular inflammation, circulatingC-reactive protein (CRP), triglycerides, and the proliferation ofvascular smooth muscle cells in the subject. Yet further, thelactoferrin composition may also increase the levels of circulating highdensity lipoproteins (HDL).

[0101] Still yet, a further embodiment is a method of enhancing immuneresponse in a subject comprising the step of administering to thesubject the composition of the present invention. It is envisioned thatthe immune response, whether local, systemic or mucosal, is enhanced bylactoferrin stimulating cytokines and/or chemokines. Exemplary cytokinesinclude interleukin-18 and GM-CSF in the gastrointestinal tract, whichare known to enhance immune cells or stimulate production of immunecells. For example, interleukin-18 enhances natural killer cells or Tlymphocytes. In specific embodiments, interleukin-18 (IL-18) enhancesCD4+, CD8+ and CD3+ cells. It is known by those of skill in the art thatIL-18 is a Th₁ cytokine that acts in synergy with interleukin-12 andinterleukin-2 in the stimulation of lymphocyte IFN-gamma production.Other cytokines or chemokines may also be enhanced for example, but notlimited to IL-12, IL-1b, MIP-3α, MIP-1α or IFN-γ. Other cytokines orenzymes may be inhibited for example, but not limited to IL-2, IL-4,IL-5, IL-10, TNF-α, or matrix metalloproteinases. It is furthercontemplated that lactoferrin inhibits the production of TNF-α, whichinhibits cells involved in inflammation: It is also envisioned thatlactoferrin stimulates interleukin-18 and a Th₁ response following oraladministration, which inhibits pro-inflammatory cytokines, e.g., IL-4,IL-5, IL-6, IL-8 and TNF-α.

[0102] The lactoferrin composition of the present invention can alsoresult in inhibition of a cytokine or chemokine. The cytokines include,but are not limited to interleukin-2 (IL-2), interleukin-4 (IL-4),interleukin-5 (IL-5), interleukin-10 (IL-10), and tumor necrosis factoralpha (TNF-α). Still further, the lactoferrin composition can alsoinhibit the production of matrix metalloproteinases (MMPs).

[0103] In further embodiments, cytokines, for example, interleukin-18 orgranulocyte/macrophage colony-stimulating factor, can stimulate theproduction or activity of immune cells. The immune cells include, butare not limited to T lymphocytes, natural killer cells, NK-T cells,macrophages, dendritic cells, and polymorphonuclear cells. Morespecifically, the polymorphonuclear cells are neutrophils and the Tlymphocytes are selected from the group consisting of CD4+, CD8+ andCD3+ T cells.

[0104] In further embodiments, the composition of the present inventionalso contains metal chelators, for example, but not limited toethylenediaminetetraacetic acid (EDTA),[ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA),1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),hydroxyethlene triamine diacetic acid, (HEDTA) or salts thereof.

[0105] Treatment regimens may vary as well, and often depend on thehealth and age of the patient. Obviously, certain types of disease willrequire more aggressive treatment, while at the same time, certainpatients cannot tolerate more taxing regimens. The clinician will bebest suited to make such decisions based on the known efficacy andtoxicity (if any) of the therapeutic formulations.

[0106] In specific embodiments, the composition is given in a singledose or multiple doses. The single dose may be administered daily, ormultiple times a day, or multiple times a week, or monthly or multipletimes a month. In a further embodiment, the lactoferrin is given in aseries of doses. The series of doses may be administered daily, ormultiple times a day, weekly, or multiple times a week, or monthly, ormultiple times a month.

[0107] D. Combination Treatments

[0108] In order to increase the effectiveness of the composition, it maybe desirable to combine these compositions and methods of the inventionwith a known agent effective in the treatment or prevention ofcardiovascular disease or disorder, for example known agents to treat orprevent atherosclerosis. In some embodiments, it is contemplated that aconventional therapy or agent, including but not limited to, apharmacological therapeutic agent, a surgical therapeutic agent (e.g., asurgical procedure) or a combination thereof, may be combined with thecomposition of the present invention.

[0109] The composition of the present invention may precede, beco-current with and/or follow the other agent(s) by intervals rangingfrom minutes to weeks. In embodiments where the composition of thepresent invention, and other agent(s) are applied separately to a cell,tissue or organism, one would generally ensure that a significant periodof time did not expire between the time of each delivery, such that thecomposition and agent(s) would still be able to exert an advantageouslycombined effect on the cell, tissue or organism.

[0110] Various combination regimens of the composition and one or moreagents are employed. One of skill in the art is aware that thecomposition of the present invention and agents can be administered inany order or combination. In other aspects, one or more agents may beadministered substantially simultaneously, or within about minutes tohours to days to weeks and any range derivable therein, prior to and/orafter administering the composition.

[0111] Administration of the composition to a cell, tissue or organismmay follow general protocols for the administration of cardiovasculartherapeutics, taking into account the toxicity, if any. It is expectedthat the treatment cycles would be repeated as necessary. In particularembodiments, it is contemplated that various additional agents may beapplied in any combination with the present invention.

[0112] A. Pharmacological Therapeutic Agents

[0113] Pharmacological therapeutic agents and methods of administration,dosages, etc. are well known to those of skill in the art (see forexample, the “Physicians Desk Reference”, Goodman & Gilman's “ThePharmacological Basis of Therapeutics”, “Remington's PharmaceuticalSciences”, and “The Merck Index, Eleventh Edition”, incorporated hereinby reference in relevant parts), and may be combined with the inventionin light of the disclosures herein. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject, and suchindividual determinations are within the skill of those of ordinaryskill in the art.

[0114] Non-limiting examples of a pharmacological therapeutic agent thatmay be used in the present invention include an antihyperlipoproteinemicagent, an antiarteriosclerotic agent, an anti-cholesterol agent, ananti-inflammatory agent, an antithrombotic/fibrinolytic agent, a bloodcoagulant, an antiarrhythmic agent, an antihypertensive agent, or avasopressor. In certain aspects of the present invention,anti-cholesterolemic agents are used in combination with the compositionof the present invention. Anti-cholesterol agents include but are notlimited to HMG-CoA Reductase inhibitors, cholesterol absorptioninhibitors, bile acid sequestrants, nicotinic acid and derivativesthereof, fibric acid and derivatives thereof. HMG-CoA Reductaseinhibitors include statins, for example, but not limited to atorvastatincalcium (Lipitor®), cerivastatin sodium (Baycol®), fluvastatin sodium(Lescol®), lovastatin (Advicor®), pravastatin sodium (Pravachol®), andsimvastatin (Zocor®). Agents known to reduce the absorption of ingestedcholesterol include, for example, Zetia®. Bile acid sequestrantsinclude, but are not limited to cholestryramine, cholestipol andcolesevalam. Other anti-cholesterol agents include fibric acids andderivatives thereof (e.g., gemfibrozil, fenofibrate and clofibrate);nicotinic acids and derivatives thereof (e.g., nician, lovastatin) andagents that extend the release of nicotinic acid, for example niaspan.Anti-inflammatory agents include, but are not limited to non-sterodialanti-inflammatory agents (e.g., naproxen, ibuprofen, celeoxib) andsterodial anti-inflammatory agents (e.g., glucocorticoids).

[0115] B. Surgical Therapeutic Agents

[0116] In certain aspects, a therapeutic agent may comprise a surgery ofsome type, which includes, for example, preventative, diagnostic orstaging, curative and palliative surgery. Surgery, and in particular acurative surgery, may be used in conjunction with other therapies, suchas the present invention and one or more other agents.

[0117] Such surgical therapeutic agents for cardiovascular diseases anddisorders are well known to those of skill in the art, and may comprise,but are not limited to, performing surgery on an organism, providing acardiovascular mechanical prostheses, angioplasty, coronary arteryreperfusion, catheter ablation, providing an implantable cardioverterdefibrillator to the subject, mechanical circulatory support or acombination thereof. Non-limiting examples of a mechanical circulatorysupport that may be used in the present invention comprise anintra-aortic balloon counterpulsation, left ventricular assist device orcombination thereof.

E. EXAMPLES

[0118] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1 Cholesterol Reduction by Recombinant Human Lactoferrin (rhLF)

[0119] Healthy adult human volunteers were administered a liquidformulation of either rhLF or placebo for seven days. Fasting serum wascollected at baseline (prior to administration of rhLF) and at the endof study (Day 10) and levels of total cholesterol determined. Allsubjects were housed in an in-patient setting for the duration of thestudy and received similar diets.

[0120] In this clinical trial, human subjects treated with placebo drugexperienced a rise in serum cholesterol. RhLF treated subjects in asimilar setting and receiving a similar diet, experienced a reduction intotal cholesterol. FIG. 1 shows that administration of rhLF for justseven days resulted in 14% reduction in total cholesterol (P<0.05).

Example 2 Reduction of C-Reactive Protein (CRP) by Recombinant HumanLactoferrin (rhLF)

[0121] Healthy adult human volunteers were administered a liquidformulation of rhLF or placebo for seven days. RhLF was administered fora total of seven days. Serum was collected on Day 1 and Day 7 of rhLFadministration and assayed for CRP using a high sensitive assay.

[0122] A total of six subjects had Day 1 CRP levels that were measurableby the high sensitive assay (>0.07 mg/dL). Five out of the six subjectsshowed a reduction in CRP with the sixth patient showing no change. Asshown in FIG. 2, the six evaluable subjects showed an average of 49%reduction in CRP levels (P<0.05) as well as a 17% reduction in theircardiovascular risk.

Example 3 RhLF Effect on Hyperlipidemia in Mice

[0123] The effect of oral rhLF was tested in a mouse model ofhyperlipidemia induced by the mice being fed a high cholesterol-fat dietfor 14 days (2 g lard, 8 g coconut oil, 1 g cholesterol, 0.3 g cholicacid per 100 g of feed and 88.7 g standard chow). The hyperlipidemicmice were administered either placebo vehicle or rhLF (1000 mg/kg) twicea day for seven days. Twenty-four hours after the last dose, serum wasobtained from individual fasting animals and assayed for totalcholesterol, HDL and LDL. The rhLF treated mice showed a trend towarddecrease in total cholesterol (16%) and LDL cholesterol (23%) and anincrease in HDL cholesterol (15%). The HDL/LDL ratio was increased by48.5% and was statistically significant (FIG. 3).

Example 4 Dose Effects of RhLF on Hyperlipidemia in Mice

[0124] Mice were rendered hypercholesteremic by administration of a highcholesterol diet for fourteen days. Animals received rhLF (32.5, 150 or500 mg/kg) or placebo administered orally twice a day for 14 consecutivedays. Twenty-four hours after the last dose of drug, fasting animalswere sacrificed and serum total cholesterol (Total, high densitylipoprotein (HDL), low density lipoprotein (LDL) and Total/HDL) ratiowere determined.

[0125] High cholesterol diet used to induce hyperlipidemia consisted of2 g lard, 8 g coconut oil, 1 g cholesterol, 0.3 g cholic acid per 100 gof feed and 88.7 g standard chow.

[0126] At the doses tested, a beneficial effect from rhLF was observedafter 14 days treatment. No dose dependence was observed. As shown inFIG. 4, all doses of rhLF decreased both total cholesterol andLDL-cholesterol relataive to placebo treated animals. A statisticallysignificant decrease in LDL-cholesterol was observed (25% decrease,p<0.05) when all the rhLF treated animals were compared to the placebotreated animals. There was also a decrease in total cholesterol (19%reduction; p=0.0594).

Example 5 Effect of Oral rhLF with Lovastatin on Hyperlipidemia in Mice

[0127] The ability of oral rhLF to potentiate the effect of lovastatinin the induced hyperlipidemia model was also tested. Hyperlipidemic micewere treated with lovastatin (15 mg/kg) alone or in combination withrhLF (500 mg/kg b.i.d.) for seven days. Mice treated with lovastatinplus lactoferrin had astatistically significant (p<0.05) reduction inboth Total Cholesterol and LDL-Cholesterol relative to the lovastatintreated mice (18% and 25% respectively) and a 13% increase in HDLcholesterol (FIG. 5).

Example 6 Reduction of Systemic Inflammation in Murine Models

[0128] Mice and rats with elevated systemic or cardiovascularinflammation are administered placebo or rhLF for 7, 14 or 28 days andmarkers of inflammation including CRP are assayed.

Example 7 Reduction of Cholesterol with rhLF Therapy

[0129] Human patients with elevated levels of cholesterol (>200 mg/dL)are administered: rhLF or placebo for 14, 28 and 90 days. Fasting serumis assayed for total cholesterol, HDL, LDL, VLDL, and triglycerides.

Example 8 Reduction of CRP with rhLF Therapy

[0130] Human patients with elevated levels of CRP are administered rhLFor placebo for 14, 28 and 90 days. Serum is assayed for CRP and othermarkers of inflammation.

Example 9 Dose Ranging Study of rhLF in the Reduction of Cholesterol andCRP

[0131] Human patients with elevated levels of cholesterol (>200 mg/dL)are given placebo or ascending doses of rhLF for 30, 90 and 180 days.Fasting serum is assayed for total cholesterol, HDL, LDL, VLDL,triglycerides and CRP.

Example 10 Reduction of Cholesterol and CRP with rhLF in CombinationTherapy

[0132] Human patients with elevated levels of cholesterol (>200 mg/dL)are administered an approved cholesterol reducing drug either alone orin combination with rhLF for 30, 90 and 180 days. Cholesterol reducingdrugs tested include Lipitor®, Zocor®, and Zedia®. Fasting serum isassayed for total cholesterol, HDL, LDL, VLDL, triglycerides and CRP.

Example 11 Reduction of Cardiovascular Incidence with rhLF Alone or inCombination Therapy

[0133] Human patients considered at an elevated risk for cardiovascularaccidents (including stroke and heart attacks) are treated with rhLFalone, approved drugs alone or a combination of rhLF and an approveddrug. Fasting serum is assayed for total cholesterol, HDL, LDL, VLDL,triglycerides and CRP. Incidence and severity of stroke and heartattacks and incidence of mortality are also measured.

REFERENCES CITED

[0134] All patents and publications mentioned in the specifications areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

[0135] U.S. Pat. No. 5,571,691

[0136] U.S. Pat. No. 5,571,697

[0137] U.S. Pat. No. 5,571,896

[0138] U.S. Pat. No. 5,629,001

[0139] U.S. Pat. No. 6,080,559

[0140] U.S. Pat. No. 5,919,913

[0141] U.S. Pat. No. 6,228,614

[0142] U.S. Pat. No. 6,455,687

[0143] U.S. Pat. No. 6,277,817

[0144] U.S. Pat. No. 6,066,469

[0145] U.S. Pat. No. 6,100,054

[0146] U.S. Pat. No. 6,333,311

[0147] Although the present invention and its advantages have beendescribed in detail, it should be understood that various changes,substitutions and alterations can be made herein without departing fromthe invention as defined by the appended claims. Moreover, the scope ofthe present application is not intended to be limited to the particularembodiments of the process, machine, manufacture, composition of matter,means, methods and steps described in the specification. As one willreadily appreciate from the disclosure, processes, machines,manufacture, compositions of matter, means, methods, or steps, presentlyexisting or later to be developed that perform substantially the samefunction or achieve substantially the same result as the correspondingembodiments described herein may be utilized. Accordingly, the appendedclaims are intended to include within their scope such processes,machines, manufacture, compositions of matter, means, methods, or steps.

What is claimed is:
 1. A method of treating a cardiovascular disease comprising the step of administering to a subject an effective amount of a lactoferrin composition to provide an improvement in the cardiovascular disease in said subject.
 2. The method of claim 1, wherein the cardiovascular disease is atherosclerosis.
 3. The method of claim 1, wherein said lactoferrin composition reduces levels of circulating total cholesterol, low density lipoproteins (LDL), very low density lipoproteins (VLDL), or triglycerides in said subject.
 4. The method of claim 1, wherein said lactoferrin composition increases the levels of circulating high density lipoproteins (HDL) in said subject.
 5. The method of claim 1, wherein said lactoferrin composition reduces the levels of vascular inflammation in said subject.
 6. The method of claim 1, wherein said lactoferrin composition reduces circulating C-reactive protein (CRP) in said subject.
 7. The method of claim 1, wherein said lactoferrin composition reduces the proliferation of vascular smooth muscle cells in said subject.
 8. The method of claim 1, wherein said lactoferrin composition reduces the vascular spasm or vascular hyper-reactivity in said subject.
 9. The method of claim 1, wherein said lactoferrin composition promotes endothelial integrity or healing in said subject.
 10. The method of claim 1, wherein said lactoferrin composition is dispersed in a pharmaceutically acceptable carrier.
 11. The method of claim 1, wherein said lactoferrin is mammalian lactoferrin.
 12. The method of claim 11, wherein said lactoferrin is human or bovine.
 13. The method of claim 1, wherein said lactoferrin is recombinant lactoferrin.
 14. The method of claim 1, wherein said lactoferrin composition comprises an N-terminal lactoferrin variant.
 15. The method of claim 14, wherein the N-terminal lactoferrin variant lacks at least the N-terminal glycine residue.
 16. The method of claim 15, wherein said N-terminal lactoferrin variant comprises at least 1% to at least 50% of the lactoferrin composition.
 17. The method of claim 1, wherein said lactoferrin composition is administered parenterally.
 18. The method of claim 17, wherein parenterally is subcutaneously, intramuscularly, intraperitoneally, intravenously, intraarterially, intramyocardially, transendocardially, transepicardially, or intrathecally.
 19. The method of claim 1, wherein said lactoferrin composition is administered orally.
 20. The method of claim 19 further comprising administering an antacid in conjunction with said lactoferrin composition.
 21. The method of claim 19 further comprising administering the lactoferrin in a delayed release formulation.
 22. The method of claim 21 where the lactoferrin release occurs in the small intestine.
 23. The method of claim 21 where the lactoferrin release occurs in the large intestine.
 24. The method of claim 1, wherein the amount of the lactoferrin that is administered is about 1 ng to about 20 g per day.
 25. The method of claim 1, wherein the amount of the lactoferrin that is administered is about 0.1 g to about 5 g per day.
 26. The method of claim 1, wherein said lactoferrin composition reduces the production or activity of pro-inflammatory cytokines.
 27. The method of claim 1 further comprising administering a lactoferrin composition in combination with an anti-cholesterol agent or an anti-inflammatory agent.
 28. The method of claim 27, wherein the anti-cholesterol agent is selected from the group consisting of cholesterol absorption inhibitors, bile acid sequestrants, nicotinic acid, fibric acids and HMG-coA reductase inhibitors.
 29. The method of claim 28, wherein the bile acid sequestrants are selected from the group consisting of cholestryramine, cholestipol and colesevalam.
 30. The method of claim 28, wherein the fibric acids are selected from the group consisting of gemfibrozil, fenofibrate and clofibrate.
 31. The method of claim 28, wherein the HMG-coA reductase inhibitors are selected from the group consisting of lovastatin, pravastatin, simvastatin, fluvastatin, atorvastatin and cerivastatin.
 32. A method of modulating atherosclerosis in a subject comprising the step of administering to said subject an effective amount of a lactoferrin composition to modulate atherosclerosis in said subject.
 33. The method of claim 32, wherein modulating is reducing the incidence of atherosclerosis in said subject.
 34. The method of claim 32, wherein modulating is reducing the severity of atherosclerosis in said subject. 